Ab51340

LV control and miR-320c HCT116 cells were lysed using RIPA buffer (Norgen-Biotek Corp.) containing 1 × Halt Protease Inhibitor Cocktail (Pierce Inc., Rockford, IL, USA). Thirty micrograms of total protein were run and blotted using the Bio-Rad V3 Western work flow system according to the manufacturer's recommendation. Immunoblotting was conducted using anti-SOX4 rabbit polyclonal antibody (H-90, dilution 1:1000, Santa Cruz Biotechnology, Santa Cruz, CA), anti-FOXM1 antibody 263C2a (ab58675, dilution 1:400), and anti-FOXQ1 antibody (ab51340, dilution 1:400), both from Abcam (Abcam, Cambridge, MA). Primary antibody was incubated overnight at 4°C. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit (cat. no. 7074, 1:3000 dilution; Cell Signaling) was used as the secondary antibody, whereas HRP-conjugated anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) antibody (ab9482, 1:10000; Abcam, Cambridge, MA, USA) was used as the loading control. Quantification of band intensity was conducted using band quantification tool in Image Lab 5.0 software (Bio-Rad, CA, USA).

Antibodies against FOXQ1 (ab51340), CD31 (ab28364), CD34 (ab81289), and F4/80 (ab6640) were purchased from Abcam (England), and paraffin-embedded mouse tumor serial sections (4 μm) were stained with anti-FOXQ1 antibody to confirm the efficiency of FOXQ1 gene knockdown. Haematoxylin and eosin (H&E) staining was performed to verify the morphological characteristics of xenograft tumor tissues. Anti-CD31 and anti-CD34 antibodies were used for EC staining. The macrophage content was measured by staining for the mature macrophage marker F4/80. For MVD analysis, CD31⁺ or CD34⁺ blood vessels in tumor sections were counted in 10 random fields (hpfs, 400×) in vascular hot spots, as previously described (30 (link)). For macrophage quantification, five random fields in F4/80⁺ hot spots were scored on a scale of 0-6 for staining intensity and distribution within a field: 0, undetectable; 1, faint, discrete patches; 2 faint, all over; 3 medium, discrete patches; 4 medium, all over; 5 intense, discrete patches; 6 intense, all over. The images were documented by two pathologists blinded and processed using Photoshop CS4 (Adobe Systems Incorporated, San Jose, CA, USA).

The slices were incubated in a pressure cooker with 0.01 M citric acid buffer (pH 6.0) for 30 min for antigen retrieval and then treated with 3% hydrogen peroxide for 5 min. Next, the slices were incubated overnight with primary antibodies, including rabbit anti-human SIRT1 (ab189494, 1:500, Abcam, Cambridge, UK), rabbit anti-human β-catenin (ab32572, 1:500, Abcam), rabbit anti-mouse β-catenin (ab16051, 1:500, Abcam), and rabbit-anti human FOXQ1 (ab51340, 1:200, Abcam) at 4°C. Diaminobenzidine (DAB) was then used for immunostaining according to the manufacturer’s instructions. The negative control (NC) group was treated with isotypic antibody. The slices were then observed under a microscope, and at least 10 high-power fields of view were selected per slice for counting of immunostained cells. The expression of SIRT1, β-catenin and FOXQ1 was semi-quantitatively scored according to the staining intensity and distribution. The samples were divided into high-expression group and low-expression group according to the median expression of FOXQ1.

Immunohistochemistry was performed with paraffin-embedded tissues cut into 2 μm sections. Tissue slides were deparaffinated by adding two times xylol for 10 min and washed with decreasing alcohol concentrations. A heat-induced epitope retrieval was performed at 100 °C and pH 8.5 in Tris-EDTA for 45 min. Afterwards, the samples were incubated with 3% H₂O₂ for 15 min followed by 5% BSA for 10 min. The antibodies were added to the tissue at room temperature as follows: anti-FOXQ1 (1:100 diluted, two hour incubation, Abcam, ab51340), anti-KRT23 (1:200 dilution, one hour incubation, Sigma Aldrich; Taufkirchen, Germany, HPA016959), anti-RIPK2 (1:50 dilution, two hour incubation, Sigma Aldrich; HPA015273) and anti-EPHX2 (1:50 dilution, one hour incubation, Sigma Aldrich; HPA035067). Visualization of the enzymatic reactivity was operated with the secondary antibody EnVision (concentrated, HRP-coupled, Dako, Hamburg, Germany) incubated for 35 min, Chromogen (DAB 1:25 diluted, ImmunnoLogic, Amsterdam, The Netherlands) for eight min and hemalaun (Thermo Scientific, Waltham, MA, USA) for two min at room temperature. The tissues were washed for five min and treated with increasing alcohol concentrations. Xylol was added for two min and the preservation performed with Vitroclud.

ChIP kit (Millipore Company) was used to study the enrichment of FOXQ1 in SIRT1 promoter region. The cells in logarithmic growth phase of each group or CRC tissues and adjacent normal tissues were added with 1% formaldehyde and fixed at room temperature for 10 min to make DNA cross-link with protein, and the complex was then randomly fragmented by ultrasonication, followed by centrifugation at 4 °C, 13000 rpm. The supernatant was collected and divided into two tubes, which were incubated overnight at 4°C with NC normal mouse IgG antibody (ab18413, Abcam) and specific anti-rabbit antibody against FOXQ1 (ab51340 Abcam), respectively. The endogenous DNA-protein complex was precipitated by Protein Agarose/Sepharose, and the supernatant was pipetted out after a short centrifugation. The non-specific complex was washed and de-crosslinked at 65 °C overnight, and the DNA fragments were extracted and purified by phenol/chloroform. The enrichment level of FOXQ1 was detected by qRT-PCR with IgG as the internal reference.

Cells were washed with PBS and lysed in ice-cold lysis buffer containing protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA) for 30 min. Lysates were separated by electrophoresis and transferred onto polyvinylidene difluoride membranes (Millipore; Bedford, MA, USA). The membranes were blocked and incubated with primary antibodies. Antibodies against FOXQ1 (ab51340), PDGF (ab178409), HB-EGF (ab92620), and Twist1 (ab175430) were purchased from Abcam (England). The antibody against EGFR (3265S) was purchased from Cell Signaling (Cold Spring Harbor, NY, USA), and antibodies against ANG (18302-1-AP), PDGFRB (13449-1-AP), ANGPT1 (23302-1-AP), PLAUR (10286-1-AP), tPA (10147-1-AP), VEGF (19003-1-AP), and β-actin (66009-1-Ig) were purchased from Proteintech (Wuhan, China). β-Actin was used as the loading control. The immunoreactive proteins were visualized with SuperSignal West Dura Chemiluminescent Substrate (ThermoFisher, Waltham, MA, USA).